Journal: bioRxiv

Article Title: Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

doi: 10.64898/2026.02.18.706588

Figure Lengend Snippet: The effect of estradiol treatment and loss-of-function mutations in neurodevelopmental disorder (NDD) risk genes is measured across three modalities: (1) single-cell RNAseq in hiPSC-derived NGN2 -induced glutamatergic neurons (iGLUTs), (2) neuronal calcium imaging in iGLUTs, and (3) sensorimotor and arousal behavior in larval zebrafish. Modalities include overlapping but independent gene sets. ASD/NDD target genes encompass myriad functions, including gene expression regulation (pink), neuronal function and communication (green), cellular signaling and protein modification (gray), and cytoskeleton (blue).

Article Snippet: H1-iCas9 hESCs were transduced with a lentivirus expressing doxycycline-inducible NGN2 (pLVX-UbC-rtTA-Ngn2:2A, Addgene #127288) modified to express blasticidin resistance in place of EGFP.

Techniques: Single Cell, RNA sequencing, Derivative Assay, Imaging, Gene Expression, Modification

Journal: bioRxiv

Article Title: Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

doi: 10.64898/2026.02.18.706588

Figure Lengend Snippet: A. Schematic of experimental design and different effect comparisons. Thirty-six NDD risk genes are targeted using pooled single-cell CRISPR-knockout screening in hiPSC-derived NGN2-glutamatergic neurons (iGLUTs) with and without 24hr exposure to 100nM estradiol. Each gene was targeted with 3-4 gRNAs (i). Various effect comparisons were performed among the treated and untreated groups (ii). Main KO effect measures KO+DMSO vs Non-targeting (NT)+DMSO. Main E2 effect measures NT+E2 vs NT+DMSO. KO effect within E2 measures KO+E2 vs NT+E2. E2 effect within KO measures KO+E2 vs KO+DMSO. E2 and KO joint effect measures KO+E2 vs NT+DMSO. B. Extent of perturbation per target NDD gene (left, dot plot). Circle size indicates negative log 10 (p-value). Stem indicates the log fold change of the knockout +DMSO vs non-targeting control + DMSO. KOs were called based on enrichment of gRNA for target NDD genes and downregulation of target KO gene expression. Number of differentially expressed genes in the main KO effect are shown in the bar plot (FDR <0.05). C . Odds of convergent genes being rescued by E2 compared to nonconvergent genes, tested across NDD KO. Fisher’s exact test, ***p<0.001, **p<0.01, *p<0.05. D. Effect of estradiol reversal on biological pathways across 12 target gene knockouts in iGLUTs. Orange color indicates that the pathway was originally downregulated in main KO effect, and estradiol upregulated the pathway in reversal effect. Blue color indicates that the pathway was originally upregulated in main KO effect, and estradiol downregulated the pathway in reversal effect. Dot size indicates the –log10 of the gene set enrichment significance of E2 effect within KO. E . Effect of estradiol rescue on DEGs across NDD risk genes in iGLUTs. Impact of estradiol on number of main KO DEGs is classified into categories of exacerbated, unchanged, partial rescue, rescue, and over-corrected. For detailed description of thresholds, see Methods. Target genes are ranked from highest to lowest percentage of DEGs that are classified as rescued (nominal p-value <0.05). F-G. Select Gene Ontology terms indicating estradiol reversal among ASH1L ( F ) and SCN2A ( G ) KO in iGLUTs. Dot size indicates the –log10 of the gene set enrichment significance of E2 effect within KO (E2) or main KO effect (KO). Asterisks for Benjamini Hochberg p-adjusted: * <0.05; **<0.01, ***<0.001.

Article Snippet: H1-iCas9 hESCs were transduced with a lentivirus expressing doxycycline-inducible NGN2 (pLVX-UbC-rtTA-Ngn2:2A, Addgene #127288) modified to express blasticidin resistance in place of EGFP.

Techniques: Single Cell, CRISPR, Knock-Out, Derivative Assay, Control, Gene Expression

Journal: bioRxiv

Article Title: Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

doi: 10.64898/2026.02.18.706588

Figure Lengend Snippet: A. Schematic of experimental workflow to assess impact of estradiol on baseline gene expression in scn1lab -/- larvae. B-C . Comparison of number of Gene Ontology (GO) pathways that are originally upregulated in HOM+DMSO and downregulated by estradiol ( B ) or originally downregulated in HOM+DMSO and upregulated by estradiol ( C ). Selected reversed pathways are highlighted in the bar graphs, with bars indicating -log10(FDR). Asterisk for the p-adjusted values: * <0.05,**<0.01, ***<0.001. D. Schematic of experimental workflow to assess impact of estradiol on baseline brain activity in HOM larvae. Dissected larval brains are stained with phosphorylated ERK (pERK) as a marker of active neurons and total ERK (tERK) as a pan-neuronal marker. Images undergo voxel-by-voxel division of pERK by tERK to obtain an activity ratio. Activity is mapped onto the standard zebrafish brain atlas and quantified based on a 4-region or 149-region atlas. E. Voxel-wise Z score normalized pERK/tERK values representing brain activity differences in scn1lab Δ44/Δ44 + DMSO or estradiol compared to DMSO-treated scn1lab +/+ larvae and the difference (subtraction) between the two conditions. Rescued regions from the 149-region atlas are shown. For specific rescued regions, refer to SI Fig. 7. Images are a representative slice of the axial (top) and sagittal (bottom) view. Scale bar represents Z score (red/yellow, increased in mutant; cyan/purple, decreased in mutant). F. Bar graphs showing regional differences in pERK/tERK brain activity in the rhombencephalon, diencephalon, mesencephalon, and telencephalon. G. Left: Schematic of experimental workflow to assess impact of estradiol on induced seizure activity in scn1lab -/- larvae. Right: Pentylenetetrazole (PTZ)-induced seizure activity, with and without 24 h exposure to 20 𝜇M estradiol, is measured in scn1lab -/- larval zebrafish mutants using video-tracking. Representative activity tracings of control and scn1lab -/- larvae at 5dpf before and after exposure to DMSO vehicle or 20uM estradiol and 10mM PTZ or water. Ribbon shows +/- SEM. H. Average activity 30 min before and after the addition of 10mM PTZ or water to control and scn1lab -/- larvae before and after exposure to DMSO vehicle or 20uM estradiol. scn1lab -/- + E2 + PTZ larvae show significantly decreased activity compared to scn1lab -/- + vehicle + PTZ larvae (Unpaired t-test with Welch’s correction on post values, ****p<0.0001; n=24/group). I. Interaction models assessing genotype-x-estradiol-x-PTZ effect, comparing post- vs pre-PTZ measurements. Dissection of the mutant effect (HOM vs control), estradiol effect (Estradiol vs DMSO), and PTZ effect (PTZ vs water) (i). Three-way interaction model to estimate the PTZ effect among genotype-x-estradiol groups of larvae (ii). J. Left: Schematic of experimental workflow to assess impact of estradiol on neuronal busting activity in CRISPR-engineered SCN2A +/- iGLUTs . Right: Cellular and network bursting activity, with and without exposure to acute 100nM estradiol, is measured in SCN2A +/- NGN2-iGLUTs using multielectrode array. Bar graphs show mean number of cellular bursts and network bursts in controls and SCN2A +/- iGLUTs. Unpaired t-test, cellular bursts *p=0.0415, network bursts SCN2A +/- spontaneous vs SCN2A +/- E2 *p=0.0127, SCN2A +/- spontaneous vs WT spontaneous *p=0.0450.

Article Snippet: H1-iCas9 hESCs were transduced with a lentivirus expressing doxycycline-inducible NGN2 (pLVX-UbC-rtTA-Ngn2:2A, Addgene #127288) modified to express blasticidin resistance in place of EGFP.

Techniques: Gene Expression, Comparison, Activity Assay, Staining, Marker, Mutagenesis, Control, Dissection, CRISPR

Journal: bioRxiv

Article Title: MEG3 Enhances Survival of Developing Human Neurons with CLCN4 -Linked Autophagy Impairment

doi: 10.1101/2025.07.16.665078

Figure Lengend Snippet: (a) Representative images of day 6 neurons after Lysotracker treatment. Lysotracker is shown in magenta, and DAPI in blue. Scale bar = 20 μm. (b) Quantification of Lysotracker signal in day 6 neurons. The relative Lysotracker intensity of CLCN4 -variant neurons compared to the WT was presented. Each dot represents one biological replicate. (n=3-4, mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test * p =0.039 for variant A; *** p =0.0007 for variant B; ** p =0.0092 for variant C) The magenta dashed line indicates the value for WT. (c) Relative acidity in acidic subcellular organelles. Each dot represents one biological replicate. (n=3, mean ± SEM, unpaired Student’s t-test; p =0.2894) (d-g) Dynamics of endo-lysosomal vesicles. RAB5, RAB7, RAB11 and LAMP1 indicates early endosome, late endosome, recycling endosome and lysosome markers, respectively. (RAB5, n=6; RAB7, n=3; RAB11, n=4; and LAMP1, n=3 or 4; mean ± SEM, unpaired Student’s t-test) (d) Representative images of a neuron with GFP-labeled vesicular organelles. TUJ1 and DAPI are shown in red and blue, respectively. Scale bar = 10 μm. (e) Relative GFP intensity expressed in vesicular organelles (* p =0.0265 for RAB5, * p =0.0158 for RAB7, * p =0.0475 for RAB11, and ** p =0.0067 for LAMP1) (f) Number of GFP-expressing vesicles in neurons. (* p =0.0186 for RAB5, * *p =0.0066 for RAB7, and p =0.9031 for LAMP1) (g) Average distance of GFP-expressing vesicles from the center of nucleus, representing the mean linear distance of puncta from the center of the nucleus in each cell. (** p =0.0011 for RAB5, * p =0.0463 for RAB7, and * p =0.0103 for LAMP1) (h) Scheme of autophagic flux analysis using tandem fluorescent protein fused to LC3. (i) Representative images of day 6 neurons expressing tandem fluorescent-tagged LC3. Red: RFP, green: GFP, white: TUJ1. Scale bar = 5 μm. (j) Quantification of the GFP-/RFP+ puncta. (n=5, mean ± SEM, unpaired Student’s t-test; * p =0.0164)

Article Snippet: For specific subcellular vesicle visualization, lentiviral constructs encoding GFP-tagged RAB5 (Addgene #134858), RAB7 (Addgene #133027), RAB11 (Addgene #134860), and LAMP1 (Addgene #134868) were utilized.

Techniques: Variant Assay, Labeling, Expressing